Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.